JPH02110139A - Hydrophobic film material modified in antistatic property, and its preparation - Google Patents

Abstract

PURPOSE: To provide a charge-mollified hydrophobic membrane material which has a coating containing fixed formal positive charged groups, by dissolving a charge-modifying agent, which consists of a specified organic polymer, in an alkali aqueous solution of an organic solvent, bringing the formed solution into contact with a hydrophobic material substrate to coat the substrate, and curing thereof.

CONSTITUTION: A charge modifying agent, which consists of an organic polymer having a polymer containing a fixed formed positive charge groups and halohydrin groups, and a molecular weight of 1000 or more (e.g. polyamine epichlorhydrin resin), is dissolved in an alkaline aqueous solution of an organic solvent. Subsequently, the solution obtained is brought into contact with a hydrophobic material substrate (example; finepore membrane of polyvinylidene fluoride) to coat the hydrophobic substrate. Then, a hydrophobic membrane material provided with a coating containing formal positive charged groups with ion exchange function and having ionic and hydrophobic both surface function is provided by curing the coated hydrophobic substrate.

COPYRIGHT: (C)1990,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a hydrophobic membrane material that has been electrostatically modified and a method for producing the same.

BACKGROUND OF THE INVENTION Microporous membranes have been shown to have a wide variety of uses. For example, many methods have been developed to produce membranes of this type. For example, U.S. Patent No. 3.876,738
The publication describes a method for producing microporous membranes by rapidly cooling a solution of a film-forming polymer in a non-solvent system. European patent application no. 0005536 describes a similar method.

A commercially available microporous membrane made of nylon, for example, is available from Ball Corporation, Glen Cope, NY under the registered trademark Ultipol N66.

Additionally, microporous membranes made of cellulose acetate, cellulose nitrate, or mixtures thereof are also widely available from a variety of suppliers. Other membranes made of polyvinylidene fluoride (PVDF) are available under the trademark Duravore® (Millipore Corporation, Bethford, Mass.). Nylon and nitrocellulose membranes are hydrophilic, while PVDF membranes are hydrophobic. However, it is also possible to coat the P V D F membrane with a material that renders it hydrophilic. These hydrophilic Durapore® membranes are also commercially available from Millivore Corporation.

For various applications (particularly filtration and polymer transfer),
It has been suggested that membrane performance may be improved by attaching ionic functionalities to the membrane surface that act to impart a fixed formal positive charge to the membrane. Charge-modified membranes of this type are suggested in US Pat. Nos. 4,512,896 and 4,673,504 for macromolecule transfer applications (eg, DNA and protein plotting). Additionally, charged modified membranes are suggested for use as filter media in US Pat. Nos. 4,1173,474 and 4,673,504. However, both of these US patents are limited to methods and uses of charge modification of hydrophilic membranes. In fact, both of the latter US patents each present unsuccessful examples of charge modification of hydrophobic membranes.

The conclusion in each of these unsuccessful US patents is that hydrophobic polymer membranes cannot be recommended for electrostatic modification by the methods described.

For example, charge-modified microporous membranes used in polymer plotting and filtration applications utilize hydrophilic membranes as starting materials.

The term "polymer plotting" as used herein refers to a method of transferring biological macromolecules, such as nucleic acids and proteins, to a seed immobilization matrix consisting of an electrophoretic gel. . Traditionally, nitrocellulose has been used as a suitable protruding matrix. Of particular importance is nucleic acid plotting, such as DNA plotting. Various DNA plotting techniques have been developed. The most common technique is called "southern plot analysis." In this technique, DNA fragments are separated by chromatographic techniques and then denatured while in a cell. Neutralize the gel and place it between blotting papers to contact the buffer reservoir. The nitrocellulose is then placed on top of the gel and the dry plotting paper is placed on top of the nitrocellulose. As the buffer flows through the gel, the DNA is eluted and binds to the nitrocellulose, thereby depositing the DNA on the nitrocellulose.
The pattern of fragments moves. The fragment pattern can then be detected by hybridization techniques using necessarily labeled nucleic acids complementary to the specific binding fragments.

Since the development of the Southern Plot technique, numerous modifications and improvements to this technique have been developed.

For example, plotting paper can be derivatized with diazobenzyloxymethyl groups to form a material commonly referred to as DBM paper, to which DNA and proteins can be covalently bonded.

Aminophenylthioether coated paper (DPT paper) activated to the diazo type can also be used to bind DNA, RNA and proteins.

Other immobilization methods use high salt or alkaline conditions in an attempt to improve DNA, RNA and protein binding.

Other attempts to improve the bonding process have focused on plotting substrates by using more hydrophilic materials, such as nylon 66, in place of dihelocellulose. In general, the above-mentioned US Pat. No. 4,512,896 and US Pat. However, these substrates also
Limited to the production of electrostatically modified hydrophilic materials. Although these materials are an improvement over nitrocellulose, DBM paper and DPT paper, their charge retention during hybridization and reuse and performance under alkaline conditions leave room for improvement.

SUMMARY OF THE INVENTION The present invention relates to charge-modified hydrophobic substrates and methods of making and using the same. More specifically, the present invention relates to a charge-modified hydrophobic microporous membrane characterized in that most of the ion exchange capacity of the material is provided by fixed formal positively charged groups. These materials exhibit a combination of ionicity and hydrophobicity that makes them highly effective for polymer adsorption applications under a variety of conditions.

In one embodiment, the fixed formal positive charge arises primarily from quaternary ammonium functional groups that form a surface coating by contacting the hydrophobic material with a solution containing a polyamine or polyamide-polyamine epichlorohydrin cationic resin. can be applied on hydrophobic surfaces. There is no need to provide a secondary charge modifier to stabilize the coating or increase the cation banding on the microporous surface.

The materials of the invention are suitable for applications such as polymer plotting and filtration. In the case of polymer plotting, a combination of hydrophobic and ionic properties is required for materials that can effectively bind macromolecules, retain them upon hybridization, and maintain signal strength over many repeated reuses. give.

The cationically charged modified substrate of the present invention consists of a hydrophobic microporous membrane that is charge modified with fixed formal charged groups having a net positive charge.

The performance of these charged modified membranes results from the combination of both hydrophobic and ionic surface effects.

As used herein, the term "microporous membrane" means an average pore size of at least 0.05 IJm, or 8,4
A substantially isotropic porous membrane having an initial bubble point (IBP) of less than 36k(]/Cm2(120r)si) is meant. The maximum pore size useful in this invention is about 10#. As used herein, the term "isotropic" or "symmetric" means that the pore structure is substantially uniform over the duration of the membrane. Additionally, "anisotropic" or "asymmetric" membranes can also be formed as composites between thin layers of material with a small average pore size supported by a more porous structure.

Membranes useful in this invention include hydrophobic polypropylene, polyethylene, polysulfone, and polytetrafluoroethylene (P
membranes made of polyvinylidene fluoride (PVDF) are preferred. PVDF membranes are described, for example, in U.S. Pat.
No. 203.847, both of which relate to Grandin two-phase, the teachings of which are incorporated herein by reference. In general, these hydrophobic membranes are
For example, it is commercially available as Immobilon P® microporous membrane (registered trademark of Millibore Corporation, Bedsford, Mass.).

The charge modifier used in the present invention is a water-soluble organic polymer having a molecular weight greater than about 1000, where the polymer consists mostly of monomers that have been reacted with epichlorohydrin. Epichlorohydrin acts to convert the tertiary amine structure previously remaining on the polymer into a structure having quaternary functionality. This forms a stable coating containing a quaternary ammonium fixed formal charge that can impart a net positive charge to the blocking substrate.

A charging modifier is coated onto the contacting surface of the hydrophobic microporous membrane. As used herein, the term "coating" refers to a charge modifier that is sufficiently bound to the membrane that it is not significantly extracted under the conditions of use.

The charge modifier is preferably a polyamine epichlorohydrin cationic resin, such as that described in U.S. Pat.
No. 116, No. 2,926.154, No. 3,224,9
No. 86, No. 3,311,594, No. 3.332.90
No. 1, No. 3.382,096 and No. 3.761, 3
No. 50, and the teachings of these US patents are cited here for reference.

The charge modifier must have a halohydrin substituent and a fixed formal positively charged group. Charge modifiers having epichlorohydrin substituents and quaternary ammonium ions are most preferred. One type of charge modifier of this type is R4
308 Resin (registered trademark of Vercules Corporation, Wilmington, PR) is a polyamine epichlorohydrin resin.

In this resin, the charged nitrogen atom is one of the heterocyclic groups.
and bond to the modified chlorohydrin group via the methylene group. Each monomer group of Vercules R4308 is represented by the following general formula; other suitable charge modifiers are Polycup 1884 [a registered trademark of Vercules Corporation, Wilmington, P.O.; However, it must be pointed out that ions other than ammonium ions, such as sulfonium, phosphonium, etc., which form fixed formal positively charged groups, may also be used in the practice of the present invention. It won't happen.

In a broad sense, the method of the present invention involves the use of materials such that the majority of the ion exchange capacity of the material is provided by fixed formal positively charged groups.
For example, it is directed to the cationically charged modification of hydrophobic organic polymeric materials such as microporous membranes. The method consists of applying to the membrane a charge modifying amount of a cationic charge modifier that coats the membrane structure and is crosslinked through halohydrin substituents and residual tertiary amines in the resin. In its broadest scope, the methods of the present invention include (a) forming a hydrophobic H material; (b) contacting said substrate with a charged modifying solution, said solution having a molecular weight greater than about 1000; The charging modifier comprises an organic polymer having a polymer chain having a fixed formal positively charged group and a halohydrin substituent, and the charging modifier is dissolved in an alkaline water-miscible organic solvent. and (c) curing the cationically charged modified hydrophobic substrate.

As mentioned above, the hydrophobic material matrix can be used in a number of hydrophobic membranes, such as polypropylene, polyethylene, polysulfone,
It can be made of any material such as PTFE. Hydrophobic P
VDF is preferred. The charge modifier is preferably a polyamine epichlorohydrin resin and is present in an aqueous solution of a water-miscible organic solvent at about 0.5 to 5% solids by weight, with the solution containing about 1 to 3% solids by weight. suitable. The organic solvent can be any lower alcohol commonly used in industrial processing. methanol, ethanol,
Isopropanol and mixtures thereof are preferred. This alcohol/water mixture has a curvature of 10 to 10% depending on the solvent.
It is a 50% by weight mixture. This mixture may contain, for example, Na
It is made slightly alkaline by the addition of substances such as OH.

Preferably, the solution has a pH of about 9.0-12.0.

Unlike other charge-modified hydrophobic membranes known in the art, the charge-modified hydrophobic membranes produced by the method of the present invention can be treated with a secondary charge modifier such as, for example, tetraethylenepentamine. Not done. Traditionally, secondary charge modifiers have been suggested as a means to increase the cationic charge of the primary charge modifier, as well as to crosslink the secondary charge modifier and improve its binding to the membrane surface. ing.

The fixed formal positively charged group used in the present invention is desirable in the sense that it provides the substrate with excellent ability to bind polymers.

When used in combination with a hydrophobic membrane, the ion exchange capacity of the fixed formal positively charged groups and the hydrophobicity of the membrane combine to provide excellent performance in applications where it is desired to create macromolecular attachment. Applications of this type include polymer plotting as well as filtration.

In plotting applications, the cationically charged modified hydrophobic membranes of the present invention can be applied to nitrocellulose, DBM paper, DP
T-paper and charged modified hydrophilic membranes are used in the same manner as conventionally used. However, the combination of hydrophobicity and fixed formal positive charge gives the membranes of the invention improved performance over currently used plot matrices.

In general, the materials of the invention have been found to be effective when used under conditions where plotting materials such as nylon have been found to be unsatisfactory (i.e., environments with high ionic strength). .

Furthermore, the materials of the invention have many advantages over conventional matrix materials such as nitrocellulose. For example, when used as a material for DNA plotting, nitrocellulose immobilized matrices require conditions of high salt concentration in buffer solutions to function satisfactorily. High salt conditions, however, require high current demands (ie, up to about 5 amps). These high currents generate heat and adversely affect the DNA being tested. Since high salt conditions are not necessary with the materials of the present invention, current can be maintained in the milliampere range and thermal damage can therefore be minimized or completely prevented.

Plots or stacking of moving electropherograms can be performed using the materials of the invention as is conventionally done using other immobilized matrix materials. Furthermore, the binding ability of the material according to the present invention is superior to that of conventional materials, so that the material according to the present invention has an improved ability to retain bound macromolecules during hybridization, as well as a strong signal during repeated reuse steps. Demonstrates ability to maintain. Although the materials of the present invention have been found to be particularly useful with respect to the transfer of macromolecules from chromatographic substrates, virtually all macromolecules of this type, including specific techniques that cause transfer to occur by convection or diffusion, It can also be used for protruding techniques.

The charged modified hydrophobic III material produced by the above method does not immediately wet when immersed in an aqueous solution. Rather, in applications where the material must be wetted before use, such as in polymeric plotting applications, it is necessary to prewet the material with the water-miscible organic solvent itself or with an aqueous solution. As mentioned above, the water-miscible organic solvent can be any lower alcohol conventionally used in the industry (ie, methanol, ethanol, isopropanol). These alcohols are preferably used as aqueous solutions with alcohol to water ratios varying from about 10 to 50% by weight, depending on the alcohol chosen.

As an alternative to the hydroalcoholic prewetting step, membrane materials used in applications requiring prewetting can also be prepared with surfactants or wetting agents incorporated into the matrix. In this case, the hydrophobic membrane is contacted with an aqueous solution of a surfactant or wetting agent during application of the charge-modifying coating or after the curing step. Suitable surfactant solutions are, for example, Tween 20 [registered trademark of ICI Inc., USA] or Pluronic F-68 [BA
Aqueous solutions of ionic or non-ionic detergents such as SF Corporation (registered trademark of SF Corporation) are included. Suitable wetting agents include tetraglyme [tetraethylene glycol dimethyl ether], glycerin, and the like. This type of treatment renders the materials of the invention wettable immediately upon contact with an aqueous medium.

[Example] Hereinafter, the present invention will be explained in more detail with reference to Examples. The following examples illustrate methods of making and using cationically charged modified hydrophobic membranes. These membranes are useful in many applications including polymer plotting and filtration.

In a broad sense, cationically charged modifiers are coated with hydrophobic organic polymers by the following procedure: the hydrophobic membrane is first prewetted in a water-miscible organic solvent (i.e. alcohol) and then water-miscible. exchange with. The membrane is then coated in an aqueous solution of a charge modifier. Alternatively, the method includes contacting the hydrophobic membrane substrate with an aqueous solution of a water-miscible organic solvent (ie, an alcohol) containing a charge modifier. Coatings can also be applied by dipping, slot coating, transfer rolling, spraying, and the like. Then,
The coated membrane is dried under restraint and cured. Suitable drying and curing methods include using an electrothermal transfer drum, hot air impingement, radiant heating, or a combination of these methods.

Example 1A: Preparation of a charge-modified microporous membrane The microporous membrane was Immobilon P®, i.e. with a pore size of 0. ,l
15 particles of hydrophobic polyvinylidene fluoride, and Durapore (
(7) 15.0x 25. A 0 cm sheet was coated with a 3% (V/V) solution of Verculece R/1308 adjusted to 1) Hlo, 56 in NaOH for 1 hour at room temperature. In the case of hydrophobic substrates, the membrane was first prewetted with methanol and exchanged with water before being placed in the coating solution. The hydrophilic Duravore was placed directly into the coating solution. After coating, the membranes were allowed to air dry overnight, then placed between sheets of polyester thin film and placed under restraint for 1 hour.
Heated at 21°C for 3 minutes.

Example 1 day: Production example 1 of charged modified microporous membrane
Membrane samples similar to A were coated with a 3 wt% solids solution of Vercules R4308 in 25% (V/V) isopropanol/water adjusted to pH 10 with 50% (W/W) NaOH. . These membrane samples were added to this solution.
Immersed for 5-1.0 minutes and then removed from the coating solution.

Excess resin solution was removed with a "squeezing" action with a wiper rod. The membrane was then dried at a temperature of 95° C. for 3 minutes in contact with an electrically heated transfer drum while being restrained between sheets of polyester thin film. Membrane samples were characterized for thickness, initial methanol bubble point, flow time, BFT surface area, and ion exchange capacity (IEC). The results are summarized in Table 1 below.

First surface film thickness, cm (mil) Initial bubble point kg/cm 2 (psi) 1 Flow time (d/min /cm2 BET surface area (m 2 /(7) IEC capacity 3 0 0.1375
．． 5 Charged modification 0.012 (4,68) 0,799 (11,36”) 30.0 Comparison 0.012 (4,65) 0,787 (11,2> 51.2 6.47 〉 1: A disk of membrane sample, 47 mm in diameter, was placed in a test holder that sealed the edges of the disk.A stainless steel support screen was placed above the membrane and in direct contact with its top surface, and the screen was sealed. The phosphorus prevented the membrane from deforming or rupturing when air pressure was applied from below. Methanol was then placed on top of the membrane. Air pressure from a controlled source was then applied from below the membrane. and increased until the first stream of bubbles is ejected by the membrane. This pressure is called the initial bubble point in cm (psi).

2: Flow time is ASTM method No. F31? /
72 i: Therefore, it was measured using the same apparatus as above.

3: To measure the total IEC capacity, a disk of 47 mm diameter membrane sample was incubated in 50% (V/V) ethanol at 0,1
100m1 of M HCe! for 5 minutes and then air dried for 1 hour at room temperature. The membrane disks were then wetted with 100 ml of an 80% (V/V) methanol/water mixture, to which was then added a 2-5M NaNO3 solution. The chloride ion concentration was then measured using a Fisher computer-assisted titrator to measure silver nitrate (0,0282 M
Estimated by inductive titration with AQNO3). Then,
The IEC capacity is expressed as the weight per millimeter of the disc/g.

Example 2: Charge Modified Microporous Membrane In this example, binding of DNA to a sample of a microporous membrane coated with a charge modifier as described in Example 1A was
Tested using Todd-Plott manifold analysis. In this assay, the membrane sample forms the bottom of a well into which liquid can be placed and incubated with the membrane.

The liquid can then be drawn through the membrane under reduced pressure. In this test, the restriction enzyme 1-1indll (N
Bacteriophage M1 cleaved with Ebiolaps
Using a sample of double-stranded DNA derived from the replication form (RF) of No. 3, the DNA binding efficiency between the charge-modified PVDF surface and nylon was compared. RF-DNA Methods in Enzymology, Vol. 10 by J. Spring.
Volume 1, Recombinant DNA [Part C], pages 20-78, R
Isolation was performed as described in 1997, edited by K., Wu, Grossman, and K1 Moldave, Academic Press, New York (1983). The DNA was transfected with the enzyme terminal deoxynucleotidyl transferase and [α-P32] as a label using a commercially available kit (NEN/DuPont, NEKOO9).
The 3'-end was labeled using 3'-dATP. 10. combining the labeled DNA with an unlabeled substance; OO
It gave the specific activity of Ocpm/1I9DNA. The DNA was then added to the wells of a dot-plot analysis manifold in a volume of 0.050 m in one of the following reagent systems: 0.125M NaOH 10.125XS
SC (standard sodium citrate, 18.75 mHN a
c l, 2.1 mH sodium citrate), 10X
SSC (1,5 M NaCe, 0.17 M sodium citrate, pH 7,4), and 25 mM sodium phosphate buffer (pH 7,4). DNA was heated to 100 °C for 5 min in the latter two buffers. later in the water 1
It was made into a single strand by rapid cooling for 0 minutes and then applied to a test membrane sample. After 30 minutes, the residual liquid was sucked through the membrane under reduced pressure.

The membrane was then placed under reduced pressure in a manifold for 0.100 d
with the same sample buffer, then the membrane sheet was removed and air-dried. Individual membrane discs were then placed in scintillation counter fluid and p32 incorporation was measured.

Other samples are described in the published literature [Molecular Cloning, pp. 387-389, Wang, Maniatis, E., F., Fritsch and J., Sampurczuk, eds., Cold Spring Harbor Press (1982)]. The retention of radiolabeled DNA was analyzed under conditions that mimic the hybridization assays used. Furthermore, the bound DNA
Also under alkaline "stripping" conditions (0.4M N
aOH, 42° C. for 30 minutes) to remove the bound radiolabeled DNA rprobe. This mimics the conditions that the membrane will experience if the plotted DNA is retested. Hydrophobic substrates and closely related hydrophilic substrates and 1
7 people DNA retention% applied in the following buffer 1 Alkaline 2 10XSSC3 Phosphate 4 Membrane type: R430B coated 100 100 70
．． 1 Hydrophobic PVDF R4308 coated 95.2 0 64.
7 Hydrophilic PVDF Nylon 5 100 95.2 61
．． 11: M1 of 1μ9 in one of the above sample buffer systems
3RF-DNA from 9.000 to 13. OOOCpm
It was applied in a volume of 0.050 m at a specific activity of ///9 DNA. After scintillation counting, μ of retained DNA
9 was calculated and expressed as % of 1μ9 of the initially applied sample.

2: 125mHN aOH, 18,75mHN
aCl.

2.1 mN citrate i~Thulium 3: 1.5 M NaCe, 0.17 M sodium citrate, DI-(7,4 4:25 mN sodium phosphate, p) (7,45: The nylon sample used was Genescreen・Plus (NEN/
DuPont).

The results of this study demonstrate that charged modification of microporous hydrophobic PVDF membrane surfaces will result in membranes exhibiting improved DNA binding and retention properties under conditions that mimic hybridization analysis and retesting. ing. The experimental conditions tested in this example are a representative range of conditions as experienced with membranes useful for in-phase nucleic acid plotting. It is particularly noted that there is no DNA binding to the R4308 coated hydrophilic PVDF surface under high ionic strength conditions (i.e. 10x S S C), which are primarily used in the case of Southern plot analysis. In contrast, hydrophobic surfaces exhibit excellent NA binding. The observed performance benefits support the theory that a hybrid ionic/hydrophobic microenvironment promotes more efficient polymer plotting. This is an important attribute of charge-modified hydrophobic surfaces.

In this example, membrane samples made as described in Example 1A were used in a manifold dot-plot analysis in a series of five cycles of hybridization.

This example demonstrates the improved performance of a charge-modified hydrophobic membrane compared to currently available nylon substrates for this application.

In this analysis, M13-RF DNA isolated as described in Example 2 was radiolabeled as follows: (1) After cutting with Hindm restriction enzyme, the sample was
A sample of complete RF-DNA was end-labeled and used to measure DNA binding as described in Example 2 under 10x S SC high ionic strength conditions, using a commercially available kit (NEN/
[α-P32]d-A by DuPont, NEKOO4)
Nick translation with TP achieved a specific activity of T 107 cpm/μh DNA, which was used as a probe for hybrid analysis. This analysis is then plotted as a dot plot.
It was carried out as described in Example 2 using a manifold (BioRad).

Two sets of DNA samples cut with HindIII (0,05
(1μ9 of 7) was adsorbed onto the test membrane as follows: (1
) Trace labeling with P323'-terminal label or NA (which was used to determine DNA retention). (2) Unlabeled DNA (which was used to perform hybridization analysis with nick translation probes). Example 2
After one cycle of hybridization as described in
While retaining a set of samples for syndesis counting,
The remaining sample was subjected to alkaline "stripping" to remove radioactive probe DNA. The latter samples were then reused in the hybridization step. This was repeated a total of 5 times. The results are shown in Tables 4 and 5. DNA binding/retention cycle model No. 1 in one cycle.

R4308 coated 1 Hydrophobic PV leaf retention rope DNA1 0.65 Nylon 3 0.26 0.20 0.21 0.17 0.22 0.018 0.034 0.027 0.014% Total 2 61.8 22.8 17.5 18.4 14.9 22.5 1.8 3.5 2.7 1.9 1: M13 RF-D cleaved with HindI[l
p32 NA to a specific activity of 15.780 cpm/iIg.
3'-terminally labeled. Sample (1 in 0,05rnI
μ9) was coupled to the membrane sample in a dot-plot manifold as described in Example 2. Then,
The membrane was removed from the manifold and cycled through hybridization analysis and [alkaline stripping] conditions to simulate a 5-cycle retest. retained DNA
The amount of was estimated by scintillation counting.

2: DN loaded into membrane sample before hybridization analysis
The comparative values of A are as follows: R4308 coated 1.14μ9 hydrophobic PVDF nylon 0.98μ93: For comparison, Genescreen Plus type 5th cycle membrane type Nα hybridized CPM1R430
8 coated 1 116.107 hydrophobic PV
DF 2 113゜3 149゜4 221゜5 113゜Nylon 2 1 32゜2
40°3 32°4 69°5 22°1: Dot-plotted M13 RF-DNA
using the established method [Molecular Cloning, No. 38]
Pages 7 to 389] was hybridized to a specific activity of T 107 Cpm/μ9 DNA. After rigorous washing, p32
Samples of probe-bound membranes (in duplicate) were subjected to scintillation counting. The degree of hybridization is λDN
Adsorbed unlabeled HindI in A [expressed as CPM bound to the cleavage product].

2: For comparison, Genescreen Plus type nylon was used.

As is clear from the data presented in Table 4, DNA
Retained well on the surface. However, in both cases, the large amount of initial DNA adsorbed on these surfaces appears to be lost during the first cycle of use and then remains at higher levels.

The hybridization analysis shown in Table 5 reflects the performance advantage of higher DNA retention with the charge-modified hydrophobic PVDF surface. Improved dot blots]
The analytical performance indicates an important attribute of the hybrid ionic/hydrophobic membrane surface.

Analysis In this example, a sample of a charge-modified hydrophobic PVDF membrane prepared by the method described in Example 1B was used in a dot-plot manifold analysis to determine the hybridization analytical performance of a charge-modified PVDF surface on nylon. compared. Batateriophage M13 RF-DNA cut with restriction enzyme 1-1indlII was added to 10x S S
C and heat denatured with IOX in a dotu l-plot manifold as described in Example 2.
Samples of charged modified hydrophobic PVDF and nylon membranes were subjected to SSC. The concentration range was as follows: 0.
32.1.6.8.0.40; 200 and 1000
nq/well 1 piece.

The dot-plotted DNA was then hybridized with a nick-translated p32-labeled probe made as described in Example 3. Examples of radioactivity photographs obtained (
Kodak XAR-5 film, 18 hour exposure with intensifying screen) not shown in FIG.

As can be seen in the photograph shown in Figure 1, the DNA dot-plotted on the charged modified hydrophobic PVDF.
The hybridization analytical performance for is superior to that seen for nylon under the conditions of this experiment. Using this PVDF surface, the plot D is as small as 8 nq.
NA can be detected radiographically. In contrast, under the same conditions, DNA could only be detected at a level of about 200 ng for the nylon membrane sample.

In the Southern blotting method, DNA fragments were separated by electrophoresis in an agarose gel, which was then transferred to a membrane support by capillary action. This method includes the following steps: (1) acid depuration to improve the mobility of large DNA molecules, (2) alkaline denaturation to make the DNA single stranded for transport, and (3) Equilibration with high ionic strength transfer buffer (10XSSC).

More specifically, Molecular Cloning, No. 387~
See page 389.

After capillary transfer to the membrane, the plotted DNA was subjected to hybridization analysis. In the hybridization method, the following steps were performed: (1) prehybridization was first performed to reduce the specific binding of the DNA to the membrane surface, and (2) the membrane was coated with a homologous p32-labeled DNA probe (the single-stranded DNA that is complementary to the DNA bound to
A) is exposed on the membrane under conditions that cause hybridization between the probe and its target DNA sequence;
and (3) excess specifically retained probe was removed by washing under conditions of increasing stringency [see Molecular Cloning, pages 387-389 for details].
.

In this example, two experiments are described: (1) λ
The DNA fragment obtained from the Hindll digest of DNA was 1) 5'-end labeled with 32dCP (kit from DuPont/NEN). The specific activity of the resulting DNA probe was determined to be 106 cpm/μa DNA T:, and a total of 1 μ9 was used in this experiment.

The retained DNA probes were visualized by exposure to X-ray film for 18 hours (Figure 2).

(2) In an experiment similar to that shown above, DNA probes were labeled to a specific activity of 109 cpm/u9DNA by using a large number of small random oligonucleotides as primers for DNA polymerase (Pharmacia kit), and sono0 .21I9 was used in the experiment.

The retained DNA probes were made visible by exposure to X-ray film for 10 minutes (Figure 3). Figure 2 compares the DNA binding ability of a nylon membrane and a charged modified hydrophobic surface.

As evident from the data (except lane 2 for the non-typical nylon), the charge-modified hydrophobic membrane gave better signal detection under the conditions of this experiment. In Figure 3, using different probes with higher specific activity,
Almost the same results are seen. The enhanced Southern Plot performance of the charged modified aqueous surface is an important property of this material compared to nylon with accepted Southern Plot technology.

Although the invention has been described in terms of preferred embodiments, those skilled in the art will recognize that many modifications may be made within the spirit and scope of the invention.

[Brief explanation of the drawing]

Figure 1 is a radiograph comparing a nylon membrane and a charge-modified microporous membrane for dot-plot hybridization analysis;・This is a radioactivity photograph diagram comparing each material in Figure 1 in plot analysis, and Figure 3 is a radioactivity photograph comparing each material in Figure 1.
This is a radioactivity photograph similar to the figure. Engraving of the drawings (no changes in content) Figure 1 Figure 2 Already Wani Shore Modified VDF DNA Filling 1111 0,56 O132 DNA Filling; 40, O05ug Figure 3 Procedure Amendment (Method) % formula % Display of the case 1999 Patent Application No. 155986 Name of the invention Charge-modified hydrophobic membrane material and its manufacturing method Amendment person Relationship with the case Patent applicant name Name
Miliboa Corporation Agent - 0,56 Address 3-13-11 Nihonbashi, Chuo-ku, Tokyo 103 Date of Notification of Amendment Order September 26, 1999 4th Street Target of Amendment Contents of Drawing Amendment As shown in the attached sheet. 1 engraving (no changes in content)

Claims (23)

[Claims] (1) A cross-linked cation characterized in that most of the ion exchange capacity of the coated substrate is imparted by fixed formal positively charged groups, thereby forming a coated substrate with both ionic and hydrophobic surface effects. A hydrophobic substrate coated with a charge-modifying material. (2) The hydrophobic substrate according to claim 1, comprising a hydrophobic microporous membrane. (3) The hydrophobic microporous membrane according to claim 2, comprising polyvinylidene fluoride, polypropylene, polyethylene, polysulfone, or polytetrafluoroethylene. (4) The fixed formal positively charged group is formed of a water-soluble organic polymer having a molecular weight greater than 1000, and the polymer has a polymer chain having a fixed formal positively charged group and a halohydrin group. film. (5) The membrane according to claim 4, wherein the halohydrin group is an epichlorohydrin group. 6. The membrane of claim 4, wherein the fixed formal positively charged group is selected from the group consisting of quaternary ammonium, quaternary phosphonium, and quaternary sulfonium. (7) The membrane according to claim 3, wherein the fixed formal positive charge is imparted by a polyaminoepichlorohydrin surface coating. (8) The membrane according to claim 1, further comprising a surfactant or a wetting agent mixed into the material. (9) A polymer analysis method comprising the steps of generating a chromatographic polymer pattern on a gel matrix, eluting the polymer pattern onto an immobilized matrix, and hybridizing the eluted substance to detect the polymer pattern. In this method, a cross-linked cationically charged modified material is used to impart most of the ion exchange capacity of the coated substrate through fixed formal positively charged groups, thereby forming an immobilized matrix with both ionic and hydrophobic surface effects. A method for analyzing macromolecules, characterized in that a coated hydrophobic substrate is used as an immobilization matrix. (10) Claim 9 wherein the hydrophobic substrate comprises a hydrophobic microporous membrane.
Method described. (11) The method according to claim 10, wherein the hydrophobic substrate comprises polyvinylidene fluoride, polypropylene, polyethylene, polysulfone or polytetrafluoroethylene. (12) The method according to claim 9, wherein the fixed formal positively charged group is formed of a water-soluble organic polymer having a molecular weight greater than 1000, and the polymer has a polymer chain having a fixed formal positively charged group and a halohydrin group. Method. (13) The method according to claim 12, wherein the halohydrin group consists of an epichlorohydrin group. (14) In producing a hydrophobic material with a coating imparting fixed formal positively charged groups, the steps of: (a) forming a hydrophobic material matrix; (b) contacting said substrate with a charge-modifying solution; The solution includes a charge modifier consisting of an organic polymer having a molecular weight greater than 1000, said polymer having polymer chains with fixed formal positively charged groups and halohydrin groups, and said charge modifier being alkaline. A method for producing a hydrophobic material, comprising dissolving it in an aqueous organic solvent solution, and (c) curing the coated hydrophobic substrate. (15) Claim 1 in which the hydrophobic substrate comprises a hydrophobic microporous membrane.
The method described in 4. (16) The method according to claim 15, wherein the hydrophobic microporous membrane is made of a material selected from the group consisting of polyvinylidene fluoride, polypropylene, polyethylene, polysulfone, and polytetrafluoroethylene. (17) The method of claim 14, wherein the charge-modifying solution is maintained at a pH of 9.0 to 12.0. 18. The method of claim 14, wherein curing comprises drying the coated hydrophobic substrate by heating. (19) The fixed formal positively charged group is quaternary ammonium,
15. The method of claim 14, wherein the method is selected from the group consisting of quaternary phosphoniums and quaternary sulfoniums. (20) The method according to claim 14, wherein the halohydrin group consists of an epichlorohydrin group. 15. The method of claim 14, further comprising the step of: (21) contacting the coated hydrophobic substrate with an aqueous solution of a wetting agent or surfactant after the curing step. (22) In preparing a hydrophobic material with a coating imparting fixed formal positively charged groups, (a) forming a hydrophobic microporous polyvinylidene fluoride matrix; (b) a 25% by volume alcohol solution in water; forming a polyamine epichlorohydrin resin, the solution containing 0.5-5% solids by weight of a polyamine epichlorohydrin resin, the solution being further adjusted to a pH of 9.0-12.0;
(d) removing the substrate from the solution; and (e) drying the substrate at a temperature of 95° C. for 3 minutes. method for producing synthetic materials. (23) A crosslinked cationically charged modified material such that most of the ion exchange capacity of the coated membrane is imparted by fixed formal positively charged groups, thereby forming a filter medium with both ionic and hydrophobic surface effects. A filtration material comprising a hydrophobic microporous membrane coated with.